The present invention relates generally to microinjection and more particularly to an electromicroinjection of macroscopic particles such as whole chromosomes into living cells.
Relatively pure chromosomes of a single type can be obtained by flow sorting. An extra copy of certain chromosomes has been shown to have biological effects due to increased gene dosage. Important questions involving gene dosage effects relative to tumorigenicity could be addressed if specific intact chromosomes could be introduced into various mammalian somatic cell types. There are several methods which have been used to alter the chromosome number of mammalian cells and study gene dosage. Fusion of two cell types, for example, relies on the gradual loss of chromosomes from the resulting hybrid cells but is often difficult to clearly interpret due to the heterogeneous nature of these hybrids. Whole chromosomes have been transferred by microcell fusion. However, many microcell hybrids have to be screened because the microcells rarely contain single chromosomes.
The most direct method for transfer of chromosomes is by microinjection. Previously reported microinjection techniques suffer from limitations in cell recovery. See, e.g., "A Microsurgical Methodology for Human Cells in Vitro: Evolution and Applications," by H. G. Diacumakos, S. Holland, and P. Pecora, Proc. Nat. Acad. Sci. 65, 911 (1970), and "Methods for Micromanipulation of Human Somatic Cells in Culture," by E. G. Diacumakos, reprinted from: Methods in Cell Biology, Vol VII, Ch. 15 (Academic Press, Inc., New York, 1973), p. 287 ff. for a description of developed procedures for manipulating chromosomes within living cells. The disclosure of the latter reference is hereby incorporated by reference herein. The former publication reports the only instance where a single chromosome was transferred from one human embryonic lung mitotic cell to another. The chromosome was dissected free from the donor cell, aspirated into a micropipette and injected into a second mitotic cell. Although the recipient cell survived the procedure and completed mitosis, it did not divide again. It was not possible to determine whether the cell was irreparably damaged by the operation, was affected by a gene on the introduced chromosome, or was incapable of growth as a single isolated cell. Clearly, a less invasive procedure would prove most valuable.
In "Microinjection of Nucleic Acids into Cultured Mammalian Cells by Electrophoresis," by M. Ocho. S. Nakai, K. Tasaka, S. Watanabe, and T. Oda, Acta Med. Okayama 35, 381 (1981), the authors describe an electrophoretic apparatus for microinjection of DNA from a conducting solution in which the DNA is dissolved into living cells. An electrode is inserted into a micropipette filled with the solution containing the molecules to be injected into the cells and placed in electrical contact with a high input-impedance preamplifier. A second electrode is placed in a conducting solution containing the cells. When the micropipette is placed in this solution an electric circuit is completed. From the impedances and the currents disclosed, one may calculate that the authors applied approximately 100 V to the microelectrode in the form of 25 ms duration, 10 Hz square waves for 5 s. No mention is made therein of the microinjection of particles not dissolved in the solution, and more particularly, no mention is made of any requirement to reverse the polarity of the applied voltage to complete the microinjection process. Moreover, the micropipette of Ocho et al. is filled in the normal manner from the end thereof away from the tip. That is, there is no teaching of attraction of the material to be injected into the cell to the tip of the micropipette by the application of a voltage thereto.
"The Introduction of Morphologically Intact Human Chromosomes into Chinese Hamster Mitotic Cells by Electromicroinjection," by F. A. Ray, W. R. Galey, J. H. Jett, and L. S. Cram, submitted for publication to Experimental Cell Research contains a more detailed account of the use of the present invention to introduce morphologically intact human chromosomes into Chinese hamster mitotic cells by the apparatus and method hereof, as does "The Introduction of Morphologically Intact Human Chromosomes into Chinese Hamster Mitotic Cells by Electromicroinjection," by F. Andrew Ray, thesis submitted in partial fulfillment of the requirements for the degree of Master of Science in Medical Sciences, The University of New Mexico, April 1985, the disclosure of the latter document hereby being incorporated by reference herein.